Method for imaging mammalian tissue using 1-substituted-1,4,7-tricarboxymethyl-1,4,7,10-tetraazacyclododecane and analogs

ABSTRACT

A method for imaging mammalian tissue utilizing a non-ionic complex of a paramagnetic ion of lanthanide element and a macrocyclic chelating agent.

This application is a continuation of Ser. No. 07/422,176, filed Oct.16, 1989, now abandoned, which is a division of Ser. No. 07/137,267,filed Dec. 23, 1987, now U.S. Pat. No. 4,885,363, which is acontinuation-in-part of Ser. No. 07/042,416, filed Apr. 24, 1987, nowabandoned, which is a continuation-in-part of Ser. No. 06/821,725, filedJan. 23, 1986, now abandoned.

Metal-chelating ligands are useful in diagnostic medicine as contrastagents. X-ray imaging, radionuclide imaging, ultrasound imaging andmagnetic resonance imaging can each be enhanced by the use of a metalatom bound to a chelating ligand. For example, a chelating ligand canbecome a radiopharmaceutical when it is prepared as a chelate complexwith ^(99m) Tc, ¹¹¹ In, ⁶⁷ Ga, ¹⁴⁰ La, ¹⁶⁹ Yb, ⁶⁸ Ga, ⁹⁰ Y, ¹⁸⁸ Re, ¹⁵³Sm or other radioactive metal ions. When a chelating ligand is complexedwith the stable isotopes of the lanthanides, tantalum, bismuth or otherelements with molecular weight higher than iodine, the resulting complexabsorbs x-rays sufficiently to act as an x-ray contrast agent. In somecases, the agents that are useful in x-ray imaging absorb, reflect orscatter ultrasound radiation sufficiently to be used as an ultrasoundagent. If a chelating ligand is complexed with a paramagnetic metal atomthat has a symmetric electronic ground state (e.g., Gd⁺³, and octahedralMn⁺², Fe⁺³, Cr⁺³) the resulting complex will be useful as a spinrelaxation catalyst that is used in magnetic resonance imaging (alsoknown as NMR imaging) as a contrast agent. If a chelating agent iscomplexed with a paramagnetic metal atom that has an unsymmetricalelectronic ground state (e.g., dysprosium(III), holmium(III) anderbium(III)), the resulting complex will be useful as a chemical shiftagent in magnetic resonance imaging or in magnetic resonance in vivospectroscopy.

The chelating ligands can also be bi-functional. That is, they can bindtightly to the metal ion forming a chelate while at the same timebearing a second functionality which confers upon it desirable chemical,physical and/or biological properties. Desirable physical properties ofthe chelator differ depending on the diagnostic or therapeutic purposeof the metal chelate. Desirable physical properties common to all usesare high affinity for the metal ion bound to the chelator and ease ofsynthesis. When it is desired to use the metal chelate as a contrastmedia for NMR imaging or general purpose X-ray imaging, the desirablephysical properties are high water solubility, and viscosity andosmolality of a formulated drug as close as possible to those of humanblood.

Human blood has an osmolality of 0.3 Osmol/kg-water. Hyperosmolality isa well known contributor to adverse patient reactions to injectedcontrast media, and the lower osmolality of newer x-ray agents is due totheir being nonionic molecules (possessing a net zero overall charge)(Shehadi, WH; "Contrast media adverse reactions: occurrence,reoccurrence and distribution patterns" Radiol. 1982, 143, 11-17.Bettman, MA; "Angiographic contrast agents; conventional and new mediacompared", Am. J. Roentgen. 1982, 139, 787-794. Bettman, MA and MorrisTW; Recent advances in contrast agents, Radiol. Clin. North Am. 1986,24, 347-357.). Gadolinium-based NMR agents in the prior art that areuseful have a net negative overall charge, and therefore their aqueousformulated solutions have high osmolality. For example, Gd(DTPA)²⁻ whereDTPA stands for diethylenetriaminepentaacetic acid is formulated for useat 0.5M in water as the N-methylglucamine salt. The osmolality of thesolution is 1.6 to 2.0 Osmol/kg-water. The preferred new gadoliniumcomplexes of the present invention are nonionic--they are not salts.When these nonionic gadolinium complexes are formulated at 0.5M in waterthe osmolality of the solutions is 0.3-0.60 Osmol/Kg-water. The complexshould be generally inert to interaction with the body other thangeneral tissue distribution and excretion, usually by the renal route.These properties are also important to NMR imaging, but, in addition,the effectiveness of an agent for NMR imaging can be increased byaltering the chemical structure so as to increase the ability of themetal chelate to affect the relaxation times of water protons.

In radiopharmaceutical imaging the doses administered are relativelysmall so that matching the drug formulation's physical properties tothose of human blood is relatively unimportant. In this use biologicalspecificity is more important. In particular, one could use ^(99m) Tc asthe metal and a chelating ligand which is functionalized with abiologically active entity such as a bile acid, fatty acid, amino acid,peptide, protein, or one of numerous chemical entities known to bindreceptors in vivo. NMR contrast media may also make use of biologicalspecificity.

In radiopharmaceutical therapy, the metal ions may be chosen from amongthose known in the art; for example, ⁹⁰ Y, ¹⁸⁸ Re, ¹⁵³ Sm. For thispurpose the chelating ligand is generally covalently bound to a diseasespecific entity such as a monoclonal antibody. When themetal-chelator-antibody conjugate is injected into humans, itconcentrates at the disease site, usually a malignant tumor. In this usethe chelating ligand must contain a reactive functionality which allowsfor a covalent bond to be formed between the chelating ligand and theantibody. Important characteristics of the reactive functionality are asfollows: 1) It must be covalently attached to the chelator such that itdoes not significantly diminish the affinity of the chelator for themetal ion. 2) It must allow simple synthesis in high yield ofmetal-chelator-antibody conjugates. The conjugate so-formed should havemaximal affinity for its antigen, such affinity being minimallydiminished as a result of covalently attaching the metal-chelator. 3) Itshould ideally allow for rapid excretion and/or optimal dosimetry of theradioactive metal chelator in the event that the metal-chelator-antibodyconjugate is decomposed or metabolized in vivo.

When the metal is non-radioactive and paramagnetic such as gadolinium(III) the bifunctional chelate is useful in magnetic resonance imagingas a contrast agent, either as a discrete molecule or bound tosubstances such as lipids, sugars, alcohols, bile acids, fatty acids,receptor-binding ligands, amino acids, peptides, polypeptides, proteins,and monoclonal antibodies. When the metal is radioactive, such ayttrium(III) as ⁹⁰ Y, the bifunctional chelate is useful in labelingmonoclonal antibodies for use in radiotherapy. When the metal is ^(99m)Tc, ¹¹¹ In, ²⁰¹ Tl, ⁶⁷ Ga, ⁶⁸ Ga or the like, the chelate is useful inradiopharmaceutical imaging.

Two general methods have been employed for making bifunctional chelatesfrom chelating agents. In the first method one or more carboxylic acidgroups of a polyamino, polycarboxylic acid chelator are activated byconversion to such activating groups as internal or mixed anhydrides,activated esters (e.g. p nitro phenyl, N-hydroxysuccinimide, etc.) orwith other derivatives known to those skilled in the art. The activatedacid group is then reacted with the protein. The metal ion is then addedto the protein-chelator complex.

There are two problems with this method. First, using a potential donorgroup, the carboxylic acid, to react with the protein can diminish thestrength of the chelate and contribute to the chemical lability of themetal ion. The second problem arises because the chelating ligands haveseveral carboxylates that are not uniquely reactive. When the chelatingligand is combined with an activating agent more than one species canresult because the number and chemical position of the groups activatedcannot be adequately controlled. When a mixture of such variouslyactivated chelating ligands is added to protein, protein-chelatorcomplexes of variable and uncertain chelating strength can be formed.Also, multiple activation of carboxylic acids on a chelator leads tointra- and inter-molecular crosslinking which is a major source ofdecreased immunospecificity. This problem could be overcome byseparating all of the products formed from the reaction of theactivating agent with the chelating ligand, but that process is verylaborious and makes the overall synthesis highly inefficient.

The second method for making a bifunctional chelate is to prepare achelating ligand with a unique reactive function, such as anisothiocyanate, attached to the chelating ligand at a position that doesnot substantially diminish the strength with which the chelating ligandbinds the metal ion. An article entitled "Synthesis of1-(p-isothiocyanatobenzyl) derivatives of DTPA and EDTA. AntibodyLabeling and Tumor-Imaging Studies" by Martin W. Brechbiel, Otto A.Gansow, Robert W. Atcher, Jeffrey Schlom, Jose Esteban, Diane E.Simpson, David Colcher, Inorganic Chemistry, 1986, 25, 2772 isillustrative of the above second method.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of this invention to provide new metal-chelatingligands.

It is an object of this invention to provide new metal chelate complexesthat are nonionic.

Another object is to provide metal chelating ligands which whencomplexed with a metal heavier than iodine (e.g. Ba, Ta, Pb, Bi,Lanthanides) are effective as X-ray contrast agents.

Another object is to provide metal chelating ligands which whencomplexed with gamma emitting radioactive nuclide (e.g. ^(99m) Tc or ¹¹¹In) are effective as imaging radiopharmaceuticals.

Another object is to provide metal chelating ligands which whencomplexed with beta or alpha emitting radioactive nuclide (e.g. ⁹⁰ Y,¹⁵³ Sm, ¹⁸⁸ Re, ²¹² Bi) are effective as therapeuticradiopharmaceuticals.

It is a further object of this invention to provide metal-chelatingligands whose metal chelate complexes in acqueous solution have lowosmolality.

It is a further object of this invention to provide metal-chelatingligands whose metal chelate complexes have low acute toxicity.

It is a further object of this invention to provide metal-chelatingligands which, when complexed with a paramagnetic metal atom, areeffective as relaxation catalysts in magnetic resonance imaging.

It is a further object of this invention to provide bifunctionalmetal-chelating ligands that have the ability to covalently bind toproteins or other biologically active molecules thereby impartingbiological specificity to the metal chelate complex.

It is a further object of this invention to provide bifunctionalmetal-chelating ligands that are themodynamically stable, kineticallyinert and, when desired, electrically neutral.

These, and other objects which will be appreciated by the practitionerof this invention, are achieved by compounds having the formula ##STR1##In formula I, and throughout the specification, the symbols are asdefined below.

Y is oxygen or ##STR2## R₁ is hydrogen, alkyl, arylalkyl, aryl, alkoxy,hydroxyalkyl, ##STR3## wherein G is NH₂, NCS, ##STR4## CO₂ H NHR₄,N(R₄)₂,CN, wherein R₄ is alkyl or hydroxyalkyl, ##STR5## wherein n and mare zero or an integer from one to five, R₂ is hydrogen or alkyl, R₃ ishydrogen, hydroxyalkyl, alkoxy, alkyl, aryl or arylkyl and X is chloro,bromo or iodo. Preferred embodiments for when the compounds are linkedto a protein are when n=2 or 1 and G=NCS.

It is understood that other functional groups known in the art can beused to link the bifunctional metal-chelating ligands of this inventionto monoclonal antibodies or fragments thereof.

R₁ and R₂ are hydrogen in a preferred embodiment for forming a Gd(III)chelate useful in general purpose magnetic resonance imaging. The mostprefered embodiment for forming a Gd(III) chelate is when R₁ ishydroxyalkyl or when R₁ is ##STR6## wherein n is 1, m is 0, G is NHR₄wherein R₄ is alkyl.

The terms "alkyl" and "alkoxy" as used throughout the specification,refer to both straight and branched chain groups. Those groups having 1to 5 carbon atoms are preferred and methyl is the most preferred alkylgroup.

The term "aryl" as used throughout the specification refers to phenyland substituted phenyl. Preferred substituted phenyl groups are thosesubstituted with 1, 2 or 3 halogen, hydroxyl, alkyl, alkoxy, carbamoylor carboxyl groups.

Hydroxyalkyl refers to straight and branched alkyl bearing radicalsR--OH groups such as --CH₂ CH₂ OH, --CH₂ CH₂ OHCH₂ OH, CH(CH₂ OH)₂ andthe like. Such chemistry is well known to those skilled in the art(Sovak, M. editor Radiocontrast Agents, Springer-Venlag, 1984 pp. 1-125.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula I, and salts thereof, can be complexed with aparamagnetic metal atom and used as relaxation enhancement agents formagnetic resonance imaging. These agents, when administered to amammalian host (e.g., humans) distribute in various concentrations todifferent tissues, and catalyze relaxation of protons (in the tissues)that have been excited by the absorption of radiofrequency energy from amagnetic resonance imager. This acceleration of the rate of relaxationof the excited protons provides for an image of different contrast whenthe host is scanned with a magnetic resonance imager. The magneticresonance imager is used to record images at various times generallybefore and after administration of the agents, and the differences inthe images created by the agents' presence in tissues are used indiagnosis. In proton magnetic resonance imaging, paramagnetic metalatoms such as gadolinium(III), and octahedral manganese (II),chromium(III), and iron(III) (all are paramagnetic metal atoms with asymmetrical electronic configuration) are preferred as metals complexedby the ligands of formula I; gadolinium (III) is most preferred due tothe fact that it has the highest paramagnetism, low toxicity, and highlability of coordinated water.

The metal-chelating ligands of formula I can be complexed with alanthanide (atomic number 58 to 71) and used as chemical shift agents inmagnetic resonance imaging or in magnetic resonance in vivospectroscopy.

While the above-described uses for the metal-chelating ligands offormula I are preferred, those working in the diagnostic arts willappreciate that the ligands can also be complexed with the appropriatemetals and used as contrast agents in x-ray imaging, radionuclideimaging and ultrasound imaging.

Use in Imaging

To use the ligands of this invention for imaging, they must first becomplexed with the appropriate metal. This can be accomplished bymethodology known in the art. For example, the metal can be added towater in the form of an oxide or in the form of a halide and treatedwith an equimolar amount of a ligand of formula I. The ligand can beadded as an aqueous solution or suspension. Dilute acid or base can beadded (if needed) to maintain a neutral pH. Heating at temperatures ashigh as 100° C. for periods up to four hours is sometimes required,depending on the metal and the chelator, and their concentrations.

Pharmaceutically acceptable salts of the metal complexes of the ligandsof this invention are also useful as imaging agents. They can beprepared by using a base (e.g., an alkali metal hydroxide, meglumine orarginine) to neutralize the above-prepared metal complexes while theyare still in solution. Some of the metal complexes are formallyuncharged and do not need cations as counterions. Such neutral complexesare preferred as intravenously administered x-ray and NMR imaging agentsover charged complexes because they provide solutions of greaterphysiologic tolerance due to their lower osmolality.

Sterile aqueous solutions of the chelate-complexes can be administeredto mammals (e.g., humans) orally, intrathecally and especiallyintravenously in concentrations of 0.003 to 1.0 molar. For example, forthe visualization of brain lesions in canines using magnetic resonanceimaging, a gadolinium complex of a ligand of formula I can beadministered intravenously at a dose of 0.05 to 0.5 millimoles of thecomplex per kilogram of animal body weight, preferably at a dose of 0.1to 0.25 millimole/kilogram. For visualization of the kidneys, the doseis preferably 0.05 to 0.25 millimoles/kilogram. For visualization of theheart, the dose is preferably 0.25 to 1.0 millimoles/kilogram. The pH ofthe formulation will be between about 6.0 and 8.0, preferably betweenabout 6.5 and 7.5. Physiologically acceptable buffers (e.g.,tris(hydroxymethyl)aminomethane) and other physiologically acceptableadditives (e.g., stabilizers such as parabens) can be present.

Use in Radiotherapy or Imaging Where the Metal-Chelate-Complex is Boundto a Biomolecule

The bifunctional metal-chelating ligands can bind to a monoclonalantibody or a fragment thereof for use in radiotherapy. Monoclonalantibodies are useful in that they can be used to target radionuclidesto cancer or tumor sites with great specificity. The compounds of thisinvention wherein R₁ is other than hydrogen are then linked tomonoclonal antibodies or fragments thereof.

The methods of linking the bifunctional chelate to the antibody orantibody fragment are known in the art (Brechbiel, same reference asreferred to hereinabove) and will depend primarily on the particularbifunctional chelate and secondarily on the antibody or fragmentthereof. For example, when the formula I compound is ##STR7## one reacts10 μl of a 5.0 mM aqueous solution of the formula I chelator with 0.5 mlof a 5.0 mg/ml monoclonal antibody (B72.3 purchaseable from DamonBiotech Corporation) in 50 mM Hepes buffer at pH 8.5. 16 μl of 1.5Maqueous triethylamine is added. After 2 hours reaction time, themonoclonal antibody is purified by dialysis. This procedure providesbetween 1 and 2 formula I chelator molecules bound to each monoclonalantibody. Radioactive metal ion (for example ⁹⁰ Y) can then be added tothe monoclonal antibody-bound chelator by methods known in the art. Forexample, ⁹⁰ Y as the ⁹⁰ Y(III)(acetate)₃ (H₂ O)₄ (approximate formula inaqueous solution) can be reacted with the mono-clonal antibody-boundchelate in solutions where the concentration of each is between 10⁻⁵-10⁻⁷ and the pH is 6. Dialysis against citrate is then used to purifythe product.

An alternative, and preferred method follows that described above, butsubstitutes the metal-chelate complex for the chelating ligand. To usethis method the metal chelate complex is first made by reactingMetal-oxide,-halide, nitrate-acetate, or the like with Formula Ichelator. For the chelator described above the acetate of ⁹⁰ Y at <10⁻⁶M is reacted with the chelator at about 10⁻³ at pH 6, the chelatecomplex is purified by ion exchange or reverse phase HPLCchromatography, and then reacted with the monoclonal antibody describedabove for the chelator. The bifunctional, metal-containing, linkedantibody is used in the following manner. A human or animal with a tumorto which the monoclonal antibody is specific is injected intravenously,subcutaneously, intraparetoneally or intralymphatically for example,with an aqueous solution of the ⁹⁰ Y-formula I chelator-monoclonalantibody compound. This allows the radioactive metal ion to be directedto the tumor for which it is intended. The intravenous dosaged used is0.1 to 0.4 millicurie per kilogram of body weight.

Preparation of Formula I Compounds

The compounds of formula I can be prepared by the reaction of a compoundhaving the formula ##STR8## with a reactive acid derivative having theformula ##STR9## wherein X is a readily displaceable group such aschlorine, bromine or iodine.

In preparing those compounds of formula I wherein Y is oxygen or##STR10## other than --NH--, the above-described reaction of a compoundof formula II with a compound of formula III is preferably carried outin water at a pH of about 9 to 10 (most preferably about 9.5). Thereaction proceeds most readily if it is warmed to about 50°-80° C. Base,such as an alkali metal hydroxide or a tetraalkylammonium hydroxide, canbe used to adjust and maintain the pH of the reaction. The reaction iscompleted in about 6 to 18 hours.

In preparing those compounds of formula I wherein Y is --NH--, theabove-described reaction of a compound of formula II with a compound offormula III is preferably carried out in water at a pH of about 8.5 to 9and the temperature of the reaction is maintained at about 45°-55° C.Preferably, only about two equivalents of a compound of formula III areinitially used in the reaction; an additional equivalent of the compoundof formula III is added in portions starting about 2 to 3 hours afterthe reaction begins. Total reaction time will preferably be about 8 to24 hours. The desired tri-substitued product can be separated from thereaction mixture, which includes the mono-, di-, tri- andtetra-substituted derivatives, by art-recognized techniques includingselective precipitation, chromatography and crystallization.

A preferred preparation of the compounds of formula I wherein Y is NHand R₂ is hydrogen is to react 1,4,7,10-tetraazacyclododecane, known inthe art, with dimethylformamidedimethylacetal in the presence of benzeneto yield 1,4,7,10-tetraazatricyclo[5.5.1.0]tridecane. This "tricyclic"compound is reacted with an ethanol/water mixture to yield1-formyl-1,4,7,10-tetraazacyclododecane. This formyl compound is thenreacted with t-butyl bromoacetate to yield 1-formyl,4,7,10-triscarboxymethyl-1,4,7,10-tetraazacyclododecane,tris-t-butylester. Finally, the ester groups are removed in the presenceof strong acid, such as sulfuric acid, to yield a compound of formula Iwherein Y is NH and R₂ is hydrogen.

Additional synthetic approaches for preparing the compounds of thisinvention will be apparent to those of ordinary skill in the art. Forexample, those compounds of formula I wherein Y is ##STR11## and R₁ isalkyl, arylalkyl, hydroxyakyl, aryl can be prepared by alkylation of thecorresponding compound of formula I wherein Y is --NH--. Those compoundsof formula I wherein Y is --NH-- can be prepared by debenzylation of thecorresponding compound of formula I wherein Y is ##STR12## and R₁ isbenzyl. The debenzylation reaction can be accomplished using catalytichydrogenolysis.

Those starting compounds of formula II wherein Y is oxygen or --NH-- areknown. The compounds of formula II wherein Y is ##STR13## and R₁ isalkyl, arylalkyl, hydroxyalkyl or aryl (this subgenus is referred tohereinafter as R'₁) are novel, and as such constitute an integral partof this invention. They can be prepared from the compound of formula IIwherein Y is --NH-- using conventional alkylation techniques.

Alternatively, the starting compounds of formula II wherein Y is##STR14## can be prepared by first reacting the 1,4,7-tritosylate ofdiethanolamine with the 1,7-ditosylate of a 4-substituted1,4,5-triazaheptane to yield ##STR15## wherein the symbol "Ts"represents the tosyl (p-toluenesulfonyl) group. This general approach topolyazamacrocycles is described in Org. Synth., 58:86 (1978). The4-substituted 1,4,7-triazaheptanes can be prepared using the methodologydescribed in U.S. Pat. No. 3,201,472.

Removal of the tosyl groups from a compound of formula IV yields thedesired compounds of formula II wherein Y is ##STR16## It can beaccomplished by acid hydrolysis using, for example, concentratedsulfuric acid or hydrobromic acid with acetic acid and phenol or byreductive cleavage using, for example, lithium aluminum hydride orsodium in liquid ammonia.

Alternatively, the starting compounds of formula II wherein Y is##STR17## can be prepared by reducing the corresponding compound havingthe formula ##STR18## using phosphorous oxychloride or phosphorouspentachloride and zinc or sodium borohydride, lithium aluminum hydride,or borane. Compounds of formula V can be prepared by cyclocondensationof diethylenetriamine with diesters of substituted imino diacetic acids,i.e., compounds of the formula ##STR19##

The compounds of formula I wherein Y is ##STR20## R₂ =hydrogen and R₁ isother than hydrogen are prepared from the compound of formula I whereinY is ##STR21## and R₂ is hydrogen namely,1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A). Thesetype of reactions are known in the art and are described below:

For convenience "DO3A" will be represented pictorially by ##STR22## inorder to illustrate the reactive secondary amine nitrogen. ##STR23## andR' and R" can be the same or different and are alkyl.

The following examples are specific embodiments of this invention.

EXAMPLE 1 4,7,10-Triscarboxymethyl-1-oxa-4,7,10-triazacyclododecane

To a solution of 8.00 g (24.8 mmol) of 1-oxa-4,7,10-triazacyclododecanesulfuric acid salt in 20 ml of water was added 6M potassium hydroxide togive a pH of 9.1. Chloroacetic acid (11.66 g, 124 mmol) was added, thepH adjusted to 9.5, and the solution warmed to 45° C. The reaction wascontinued for 15 hours with base added as necessary to maintain the pHbetween 9.5-10. The solution was cooled to 21° C., the pH brought to 2.0with concentrated hydrochloric acid, and the solution evaporated todryness. The residue was extracted with 400 ml of ethanol, filtered, andthe solvent evaporated. The solid was dissolved in water and passed ontoa cation exchange column (Dowex 50X2, hydrogen form). The column waswashed with water and the ligand eluted with 0.5M ammonium hydroxide.The solvent was evaporated, the solid redissolved in water and passedonto an anion exchange column (AG1-X8, formate form). The column waswashed well with water, and the ligand eluted with 0.5M formic acid. Thesolvent was evaporated under reduced pressure, the solid redissolved inwater and reevaporated. The crude solid was dissolved in methanol andslowly precipitated by the addition of acetone and cooling to about 5°C. The yield was 2.66 g of an extremely hygroscopic and deliquescentsolid. ¹³ C NMR (D₂ O, ppm vs TMS): 175.4, 170.7, 65.3, 58.4, 56.0,54.0, 53.7, 49.9. Mass spectrum (FAB): m/e 348 (M+H) and 346 (M-H).

EXAMPLE 2 1,4,7-Triscarboxymethyl-1,4,7,10-tetraazacyclododecane MethodI

A solution of 36.8 g (0.100 mol) 1,4,7,10-tetraazacyclododecanebissulfuric acid salt in 166 ml deionized water was brought to pH 8.5using 6M potassium hydroxide. To this solution was added 18.9 g (0,200mol) of solid chloroacetic acid, and the pH was readjusted to 8.5. Thetemperature was increased to 50° C. and the pH maintained between8.5-9.0 by the addition of 6M potassium hydroxide as necessary. After 3hours, an additional 4.73 g (0.050 mol) of chloroacetic acid was added,and the pH readjusted. After 5 hours, an additional 3.78 g (0.040 mol)of chloroacetic acid was added and the pH was readjusted. The reactionwas continued at 50° C. and pH 8.5-9.0 for 16 hours after the secondaddition. The reaction mixture was cooled, the pH brought to 2 withconcentrated hydrochloric acid, and the mixture diluted with methanol.The mixture was filtered and the filtrate evaporated. The solid wasdissolved in water and passed onto a cation exchange column (Dowex50X2-400, hydrogen form). The column was washed well with water then theligand brought off by eluting with 0.5M ammonium hydroxide. Evaporationgave the solid ammonium salt. This salt was dissolved in water andpassed onto a column of anion exchange resin (Dowex AG1-X8). The columnwas washed well with water and the ligand eluted with 0.5M aqueousformic acid. The solid obtained after evaporation of the solvent wascrystallized from methanol to give 8.2 g of the ligand as a colorlesssolid. ¹³ C NMR (D₂ O, ppm vs TMS): 176.9, 171.0, 57.0, 55.7, 52.7,50.3, 49.3, 43.6. Mass spectrum (FAB): m/e 345 (M-H) and 347 (M+H).

Method II

A mixture of 100 mg of 10% palladium on charcoal and 250 mg of1-benzyl-4,7,10-triscarboxymethyl-1,4,7,10-tetraazacyclododecane in 40ml of 5% acetic acid in water was shaken under 35.8 p.s.i. of hydrogenfor 16 hours. Filtration and evaporation gave the crude ligand which wascrystallized from methanol/acetone yielding 130 mg of the desiredproduct.

EXAMPLE 31-Methyl-4,7,10-triscarboxymethyl-1,4,7,10-tetrazacyclododecane

To a solution of 250 mg (0.723 mmol) of1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane in 2.9 ml ofmethanol was added 220 mg (1.59 mmol) of potassium carbonate. To theresulting mixture was added 308 mg (2.17 mmol, 3 equiv) of methyliodide. Within a short time, most of the solids dissolved. After 15hours at 21° C., a mass of crystals had separated. Additional methanolwas added (2 ml) to dissolve the solid. After 23 hours, an additional102 mg (0.72 mmol) of methyl iodide was added. After an additional 16hours, the solution was acidified with concentrated hydrochloric acidand the volatiles were removed on the rotary evaporator. The residue wasextracted with methanol, filtered, and the methanol evaporated. Theresidue was crystallized twice from methanol/acetone yielding 56 mg(0.16 mmol) of a colorless solid, melting point 215°-240° (dec.). ¹³ CNMR (D₂ O, ppm vs TMS): 177.2, 171.1, 57.2, 56.5, 54.1, 52.6, 50.1,49.9, 43.7. Mass spectrum (FAB): m/e 359 (M-H) and 361 (M+H).

EXAMPLE 41-Benzyl-4,7,10-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

A) 5-Benzyl-2,8-dioxo-1,5,9-triazanonane

To a solution of 6.80 g (95.7 mmol) acrylamide (95.7 mmol) in 10 mlwater at ca. 5° C. was added dropwise 4.4 ml (4.32 g, 40.3 mmol) ofbenzyl amine. After the addition was complete, the temperature wasraised to 87° C for 6 hours. The water was evaporated to give a thickoil. The oil was dissolved in about 25 ml acetone and about 15 ml ofether was added to precipitate an oil. This mixture was allowed to standfor 16 hours, during which time the oil solidified. The solid was brokenup and collected by filtration and dried under vacuum at 50° C. for 6hours. The crude product weighed 9.75 g (39.1 mmol), melted at 103°-106°C., and was suitable for use in the next reaction without furtherpurification.

B) 4-Benzyl-1,4,7-triazaheptane

To a solution of 38.7 g (0,586 mol) potassium hydroxide in 150 ml waterat 5° C. was added 30.0 g (0.120 ml) of5-benzyl-2,8-dioxo-1,5,9-triazanonane. To the resulting mixture wasadded dropwise 345 ml of 0.80M potassium hypochlorite over 0.5 hours.The solution was allowed to warm to 21° C. then heated to 85° C. for 4hours. The solvent was then evaporated under reduced pressure and theresidue was extracted with dichloromethane, filtered, dried withmagnesium sulfate, filtered, and evaporated to give 14.7 g of the crudeproduct. Vacuum distillation gave 10.1 g of the desired triamine as acolorless liquid, boiling point 110°-115° C. at 0.35 mm. of Hg. ¹³ C NMR(CDCl₃, ppm vs TMS): 139.1, 128.7, 128.1, 126.9, 59.0, 56.3, 39.3. Massspectrum (CI): m/e 194 (M+H) and 192 (M-H).

C) 4-Benzyl-1,7-bis(p-toluenesulfonyl)-1,4,7-triazaheptane

To a solution of 141.2 g (0.741 mol) of p-toluenesulfonyl chloride in250 ml of dichloromethane with 110 ml (0.8 mol) of triethylamine wasadded dropwise 68.0 g (0.352 mol) of 4-benzyl-1,4,7-triazaheptane in 75ml of dichloromethane. After 2 hours, the solution was washed threetimes with water at pH 9, and the organic phase was dried with sodiumsulfate and filtered. Evaporation gave an oil which was dissolved inabout 450 ml of ethyl acetate. The solution was diluted with 200 ml ofether and left to stand at room temperature for 24 hours. The mixturewas further diluted with about 50 ml of ether and refrigerated foranother day. The product crystallized in massive prisms which werecollected by filtration and dried under vacuum at 40° C. for 6 hours,yielding in the first crop 140 g (0.279 mol) of a colorless solid;melting point 87°-91° C. Mass spectrum (CI): m/e 502 (M+H) and 500(M-H).

D)1-Benzyl-4,7,10-tris(p-toluenesulfonyl)-1,4,7,10-tetraazacyclododecane

Into a dry flask under nitrogen was placed about 3.9 g of a 60% sodiumhydride dispersion. It was washed twice with hexanes then suspended in200 ml of dry dimethylformamide. To the mixture was added 20 g (40 mmol)of 4-benzyl-1,7-bis-(p-toluenesulfonyl)-1,4,7-triazaheptane over 5minutes. After the initial reaction had subsided, the mixture was heatedto 110° C. for 1 hour. To the resulting hot solution was added dropwise22.6 g (40 mmol) of diethanolamine tritosylate in 100 ml of drydimethylformamide over 3.5 hours. After an additional 0.5 hours, thesolution was allowed to cool and 20 ml of methanol was added. Thevolatiles were then removed on the rotary evaporator. The residue wasdissolved in a mixture of 400 ml of water and 200 ml of dichloromethane.The phases were separated and the aqueous phase washed twice more withdichloromethane. The combined organic fractions were dried (magnesiumsulfate), filtered, and evaporated to give a yellow oil. Crystallizationwas induced by the addition of about 100 ml of methanol. The mixture waskept at -5° C. overnight and the product collected by filtration. Afterdrying, 20.4 g of a colorless solid was obtained; melting point208°-210° C.

E) 1-Benzyl-1,4,7,10-tetraazacyclododecane tetrahydrochloride

Method I

To a slurry of 2.0 g (2.8 mmol)1-benzyl-4,7,10-tris(p-toluenesulfonyl)-1,4,7,10-tetraazacyclododecanein about 25 ml of ammonia at -77° C. under nitrogen was added 0.50 g (22mmol, 8 equiv) of sodium metal in portions over about 5 minutes. Theblue mixture was stirred an additional 45 minutes and the reaction wasquenched with 1.16 g (22 mmol) of solid ammonium chloride. The ammoniawas allowed to evaporate. Water (50 ml) was added to the residue and thepH adjusted to about 12 using 6M potassium hydroxide. The mixture wasextracted three times with 30 ml portions of dichloromethane. Thecombined organic fractions were then extracted with three 30 ml portionsof 2M hydrochloric acid. Evaporation of the water under reduced pressuregave a solid residue. The residue was washed with methanol and driedunder vacuum at 50° C. to give 600 mg (1.47 mmol) of a colorless solid,which was used directly in the final step.

Method II

1. 1-Benzyl-3,11-dioxo-1,4,7,10-tetraazacyclododecane

To a solution of 31.2 g dimethyl-N-benzyliminodiacetate in 2.5 l of dryethanol at reflux under nitrogen was added dropwise 12.8 g ofdiethylenetriamine in 160 ml of dry ethanol. Reflux was carried out fora total of 137 hours. The solution was evaporated under reduced pressureleaving a yellow paste. Trituration with acetone left 5.2 g of thedesired product as a colorless solid.

¹³ C NMR (methanol, ppm vs TMS): 173.6, 139.0, 130.5, 129.6, 128.8,64.0, 46.3, 38.7. Mass spectrum (CI): m/e 291 (M+H) and 289 (M-859 H).

2. 1-Benzyl-1,4,7,10-tetraazacyclododecane

To a suspension of 890 mg (3.07 mmol) of1-benzyl-3,11-dioxo-1,4,7,10-tetraazacyclododecane in tetrahydrofuranunder nitrogen was added 2.64 ml of 8M borane-methyl sulfide complex(21.1 mol, 7 equivalents). The mixture was heated to reflux allowing themethyl sulfide to distill out of the reaction flask. After 2 hours, thereaction was quenched by the addition of 12 ml 1.8M hydrochloric acid inmethanol and refluxed for an additional 3 hours. Volatiles were removedby evaporation, and the solid resuspended in methanol and reevaporated.The product was crystallized from methanol/ethyl acetate; yield 455 mg,37%. Mass spectrum (CI): m/e 263 (M+H).

F) 1-Benzyl-4,7,10-triscarboxymethyl-1,4,7,10-tetraazacyclododecane

The pH of a solution of 3.5 g of 1-benzyl-1,4,7,10-tetraazacyclododecanetetrahydrochloride in 17 ml of water was adjusted to 7 using 6.0Mpotassium hydroxide. To this solution was added 3.64 g of chloroaceticacid, and the pH was re-adjusted to 9.5. The solution was warmed to 45°C. and the pH adjusted as necessary to maintain the pH at 9.5-10. After6 hours, the heat source was removed and the solution left to stand for1 day. The solution was acidified to pH 3 with concentrated hydrochloricacid, diluted with 500 ml of water, and applied to a Dowex 50X-2 cationexchange resin (H⁺ form). After washing with water, the ligand waseluted with 0.5M aqueous ammonia. After evaporation of the solvents, thecrude ammonium salt was redissolved in water and applied to an anionexchange column. After washing with water, the ligand was eluted with0.2M aqueous formic acid. After evaporation of the solvents, the crudeproduct was crystallized from methanol/acetone to give 2.0 g of theligand as a colorless solid. Mass spectrum (FAB): m/e 437 (M+H) and 435(M-H).

EXAMPLE 5Gadolinium(III)(1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane)Method I

To a solution of 9.05 g (26.1 mmol) of1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane (see Example 3)in 50 ml of water was added 4.74 g (13.1 mmol) of solid gadoliniumoxide. The mixture was heated to 90° C. for 4 hours, during which timemost of the solid dissolved. The mixture was filtered and the filtrateevaporated to dryness under reduced pressure. The gummy residue wastwice dissolved in ethanol and evaporated to dryness. The colorlesssolid residue was dissolved in nitromethane, filtered through a fineporosity sintered glass funnel, and the filtrate placed in a flask in aclosed container also holding about 1 liter of water. Diffusion of waterinto the organic solution over several days gave a colorless solidprecipitate. The precipitate was collected by filtration, washed withnitromethane, resuspended in acetone and washed well with that solvent,then dried under vacuum at 60° C. for 2 days yielding 10.7 g of acolorless solid.

Anal. Calcd. for 90.06% ligand, 9.94% water; C, 30.25; H, 5.28; N,10.08. Found: C, 30.25; H, 5.48; N, 9.97; C/N=14.4

Method II

Gadolinium acetate tetrahydrate (145.5 mg) was dissolved in 3 ml ofdeionized water. Aqueous 1M1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane was added to thegadolinium acetate solution, mixed and adjusted to pH 3. The mixture washeated for 20 minutes at 88° C. and adjusted to pH 7.3 with 1N sodiumhydroxide. The free gadolinium content was measured by paper thin layerchromatograhy. Twice the quantity of1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane required tochelate any free gadolinium was added. The solution was adjusted to pH3, heated at 88° C. for 20 minutes and then adjusted to pH 7.3. Freegadolinium content was determined and1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane was added asrequired. The sample was adjusted to 7 ml with deionized water, passedthrough a 0.22μ filter (Millipore) into a vial, stoppered and sealed.

EXAMPLE 6Gadolinium(III)(4,7,10-triscarboxymethyl-1-oxa-4,7,10-triazagyclododecane)

Thirty mg of 4,7,10-triscarboxymethyl-1-oxa-4,7,10-triazacyclododecane(see Example 1) was added to 0.7 ml of 100 mM gadolinium acetate. Thesolution was adjusted to pH 3 and heated at 88° C. for 20 minutes. Aprecipitate was visible when the solution was adjusted to pH 7.3.4,7,10-Triscarboxymethyl-1-oxa-4,7,10-triazacyclododecane (16 mg) wasadded, the solution adjusted to pH 3 and heated at 88° C. for 20minutes. On adjustment to pH 7.3, a slight precipitate was observed.Twenty mg of 4,7,10-triscarboxymethyl-1-oxa-4,7,10-triazacyclododecanewas added and the solution was adjusted to pH 3.0. Reheating under thesame conditions resulted in reducing the free gadolinium to 0.22±0.18%as measured by paper thin layer chromatography of a radiolabeled chelatesolution. The final chelate solution was clear at pH 7.3. It was passedthrough a 0.22μ filter (Millipore) into a vial and sealed.

EXAMPLE 7 ^(99m)Technetium(1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane)

100 mM of calcium (II) (1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane) was prepared by mixing equal volumes of200 mM of calcium chloride and 200 mM of1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane. One and one-halfml of the solution was adjusted to pH 8.8 with dilute sodium hydroxideand 150 μl of 0.88% stannous tetrahydrochloride chloride was added andmixed. Technetium-99m was added to obtain a final concentration of 20μCi/ml and the solution was adjusted to pH 3. The solution was heated at88° C. for 20 minutes, cooled, and adjusted to pH 7. After adjusting toa volumne of 3 ml, it was passed through a 0.22μ filter.

EXAMPLE 8Gallium(III)(1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane)

(1,4,7-Triscarboxymethyl-1,4,7,10-tetraazacyclododecane (69.3 mg) and58.8 mg of dihydrated calcium chloride were mixed in water to yieldcalcium (II) (1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane).90 μCi of ⁶⁷ -gallium was added. The solution was adjusted to pH 3,heated at 88° C. for 20 minutes and adjusted to pH 7.3.

EXAMPLE 9Bismuth(III)(1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane)

50 mM Bismuth(III)(1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane) was prepared bycombining 24.2 mg of bismuth nitrate with 100 μl of 1M1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane and 140 μl ofacid. The solution was adjusted to pH 3 with dilute sodium hydroxide.The mixture was heated at 88° C. until the bismuth nitrate dissolved(ca. 30 minutes). The solution was adjusted to pH 7.3 with dilute sodiumhydroxide and reheated briefly at 88° C. until a small quantity ofprecipitate was dissolved. On cooling, the solution remained clear.

Determination of free bismuth by precipitation and x-ray fluorescencespectroscopy showed that >99% of the bismuth had been chelated.

EXAMPLE 10 Chromium, Iron, Manganese and Dysprosium Chelates of(1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane)

Four hundred fifty μl of 100 mM solutions of each of chromic chloride,ferric chloride, manganese chloride and dysprosium chloride were mixedwith 50 μl of 1M 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecaneand adjusted to pH 4.5. The solutions were heated at 88° C. for 20minutes to enhance the rate of chelation, cooled and then adjusted to pH7.

To determine if chelation had occurred, the solutions were diluted to aconcentration of 1 mM metal chelate. An aliquot was tested by measuringits relaxivity and comparing it with the relaxivity of the metal ionalone. The data demonstrated clearly that the metal ions had beenchelated. The relaxivity is proportional to the number of watermolecules bound to the metal. The chelator displaces coordinated watermolecules and thus lowers the relaxivity. Relaxitives of metal chelatesare shown in the following table.

    ______________________________________                                        Relaxivities of Metal Chelates at 20MHz                                       Chelate                 K.sub.1                                               Metal                   (Mole.sup.-1 Sec..sup.-1)                             ______________________________________                                        Dysprosium(III) (1,4,7-triscarboxymethyl-                                                             177                                                   1,4,7,10-tetraazacyclododecane)                                               Dysprosium chloride      525                                                  Iron(III)(1,4,7-triscarboxymethyl-                                                                     530                                                  1,4,7,10-tetraazacyclododecane)                                               Ferric chloride         3374                                                  Chromium(III)(1,4,7-triscarboxymethyl-                                                                 422                                                  1,4,7,10-tetraazacyclododecane)                                               Chromic chloride        3270                                                  Manganese(III)(1,4,7-triscarboxymethyl-                                                               1151                                                  1,4,7,10-tetraazacyclododecane)(sodium                                        salt)                                                                         Manganese chloride      6250                                                  ______________________________________                                    

EXAMPLE 11Gadolinium(III)(4,7,10-triscarboxymethyl-1-methyl-1,4,7,10-tetraazacyclododecane)

Gadolinium acetate tetrahydrate (102 mg) was mixed with 133 mg of4,7,10-triscarboxymethyl-1-methyl-1,4,7,10-tetraazacyclododecane. To themixture was added 250 μCi of ¹⁵³ gadolinium nitrate. The solution wasadjusted to pH 3 with 1N hydrochloric acid and heated for 20 minutes at88° C. The solution was adjusted to pH 7. Free gadolinium content was5.07%. Additional ligand, 36 mg, was added, the solution was adjusted topH 3 and heated as before. The solution was adjusted to pH 7.3 andtested by the thin layer chromatography procedure. Free gadoliniumcontent was 0.14%.

EXAMPLE 12 50 mMYttrium(III)(1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane).

145.4 mg of Yttrium acetate tetrahydrate Y(OAc)₃ (H₂ O)₄ is dissolved in3 ml of deionized water. 0.1 mCi of a radioactive tracer, ⁹⁰ Y inhydrochloric acid, is added. 385 μl of aqueous 1M1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane is added to theyttrium acetate solution, mixed and adjusted to pH 3 to 4 with 1Nhydrochloric acid or 1N sodium hydroxide. The mixture is heated for 20minutes at 88° C. and adjusted to pH 7.3 with 1N sodium hydroxide. Theunreacted yttrium is measured by paper thin-layer chromatography. Thequantity of 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecanerequired to react with any unreacted yttrium is added by weighing theproper amount of solid1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane or by adding anadditional volume of the 1M1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane solution. Thesolution is adjusted to pH 3 to 4, heated at 88° C. for 20 minutes andthen adjusted to pH 7.3. The process of detecting unreacted yttrium andadding further aliquots of1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane is repeated untilthe unreacted yttrium level is less than 0.05 mM as determined by theTLC method. The sample is adjusted to 7 ml with deionized water, passedthrough a 0.22μ filter (Millipore) into a vial, stoppered and sealed.

EXAMPLE 13 [⁹⁰Yttrium](III)1,4,7-(triscarboxymethyl-1,4,7,10-tetraazacyclododecane).

10 mCi of ⁹⁰ Y in a minimum volume of [0.1M] hydrochloric acid istreated with sodium hydroxide using a micropipet until the pH is 3 to 4.μl aliquots of 1M 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecaneat pH 3.5 are added to make the mixture 10⁻⁵ M in1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane and then themixture is heated for 20 minutes at 88° C. and adjusted to pH 7.3 withconcentrated sodium hydroxide. The percentage of ⁹⁰ yttrium isdetermined by thin layer chromatography. If more than 0.1% of theyttrium is unreacted, the pH is lowered to 3 to 4 and the mixture againheated at 88° C. for 20 minutes. This procedure is repeated until eitherthe level of unreacted ⁹⁰ Y is less than 0.1% of the total, or the levelis the same after two consecutive heating cycles. If the level ofunreacted ⁹⁰ Y is greater than 0 1% and not decreasing after two heatingcycles, and additional aliquot of1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane is added to makethe concentration 2×10⁻⁵ M in1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane. The heatingprocedure is then repeated until the level of unreacted ⁹⁰ Y is lessthan 0.1%.

The sample is adjusted with deionized water to the desired activitylevel, and passed through a 0.22μ filter (Millipore) into a vial andsealed.

EXAMPLE 14Yttrium(III)1,4,7-(triscarboxymethyl-1,4,7,10-tetraazacyclododecane fromY₂ O₃.

9 grams (26 mmol) of1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane in 50 ml ofdistilled water is treated with 2.95 grams of (13 mmol) Y₂ O₃. Themixture is heated at 88° C. for 4 hours, while the solid dissolves. Thesolution is filtered to remove any undissolved solid and the solvent isremoved by evaporation. Vacuum drying is used to obtain a dry solid.Alternatively, the filtered reaction solution may be spray dried.

EXAMPLE 151,4,7-Triscarboxymethyl-10-(2'-cyanoethyl)-1,4,7,10-tetraazacyclododecane

Into a 50 ml round bottom flask was placed 5.22 g (0.0151 mol) of1,4,7,-triscarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A) anddissolved in 21 ml of water. The pH of the solution was raised to 8.28with 6N NaOH. Then 1.35 ml (1.09 g, 0.0205 mol) of acrylonitrile wasadded and the reaction allowed to stir overnight at room temperature.The reaction is then concentrate in vacuo, and then re-dissolved inmethanol and concentrated in vacuo.

EXAMPLE 161,4,7-Triscarboxymethyl-10-(2'-carboxyethyl)-1,4,7,10-tetraazacyclododecane

The crude product of a 0.015 mol preparation of Example 15 was added to100 ml of 3N NaOH (large excess) and heated to 85° C. and allowed tostir under nitrogen for five hours. The inorganic salts are then removedvia cation/anion exchange chromatrography as described in Example 2,Method I.

EXAMPLE 171,4,7-Triscarboxymethyl)-10-(3'-aminopropyl)-1,4,7,10-tetraazacyclododecane

0.5 g of the crude reaction product of Example 15 was dissolved in 25 mlof water and to this was added 1 ml of conc. HCl. This solution was thenadded to 0.25 g of 10% Pd/C and then hydrogenated at approximate 40 psiovernight. The cataylst was then filtered over a Celite bed and thesolution concentrate in vacuo.

EXAMPLE 18 1,4,7-Triscarboxymethyl-1,4,7,10-tetraazacyclododecane (DO3A)

A) 1,4,7,10-tetraazatricyclo[5.5.1.0]tridecane Reagents

a. 1,4,7,10-tetraazacyclododecane 250 g (1.45 moles)

b. Benzene (sieve-dried) 2.5 liters

c. Dimethyl formamidedimethylacetal 173 g (1.45 moles)

The above were combined and heated in an oil bath at 80° C. undernitrogen while the benzenemethanol azeotrope (64° C.) distilled off.After ninety minutes, the temperature of the distillate rose to 80° C.indicating complete reaction. Distillation of benzene was continued foran additional thirty minutes to ensure complete reaction. The reactionmixture was concentrated in vacuo (50° C.) then the residue distilled(bath temperature 160° C.) to yield 253 g (96%) of desired product. b.p.128°-130° C./0.5 mm.

B) 1-formyl-1,4,7,10-tetraazacyclododecane Reagents

    ______________________________________                                        a.    1,4,7,10-tetraazatricyclo[5.5.1.0]tri-                                                                246    g                                              decane                                                                  b.    Absolute ethanol        500    ml                                       c.    H.sub.2 O               500    ml                                       ______________________________________                                    

The product of A was chilled in an ice bath (4° C.) then treated withethanol-water (pre-mixed) which had been chilled to -20° C. The mixturewas allowed to slowly warm to room temperature then stirred undernitrogen for twenty-four hours at ambient temperature. The reactionmixture was concentrated in vacuo. The residue was dissolved inacetonitrile (1,000 ml) then concentrated in vacuo. This operation wasrepeated three times (3×1,000 ml) to remove all traces of water. Theresidue was dried in vacuo at room temperature overnight. After fourhours the material crystallized with significant heat ofcrystallization. Yield 270 g (100%).

C)1-formyl,-4,7,10-triscarboxymethyl,-1,4,7,10-tetraazacyclododecane,tris-t-butylester

To the product of B, dissolved in dimethylformamide, was added 4equivalents of t-butyl bromoacetate. An initial exotherm was controlledby ice bath cooling, and after 30 minutes a solution of sodium carbonatewas added. After agitating this mixture briskly for an additional 30minutes, toluene is added and the reaction is allowed to proceed at 30°C. until complete by TLC.

After agitation is stopped, the layers are allowed to settle and thelower aqueous layer, containing mainly DMF and salts, is withdrawn.Further extraction of the toluene layer with aqueous sodium carbonateeffects removal of any remaining DMF. The toluene solution is treatedwith 1 equivalent of dilute HCl to extract the intermediate formyltriester into water, and to separate excess t-butyl bromoacetate, whichremains in the toluene layer.

Methylene chloride is added to the acidic aqueous layer, and sodiumcarbonate is added slowly as the mixture is rapidly agitated. Once asolution pH of 9.5 is reached, agitation is stopped and layers areallowed to form. The lower, rich methylene chloride solution iswithdrawn. Additional methylene chloride is added to extract theremaining aqueous layer, and the combined organic layers are thenbackwashed with fresh deionized water. The methylene chloride solutioncontaining formyl triester is concentrated.

D) 1,4,7,-Triscarboxymethyl-1,4,7,10-tetraazacyclododecane

The methylene chloride concentrate from above is added over 30-40minutes to 2 equivalents of sulfuric acid in water, maintained at55°-60° C. under a vigorous nitrogen sparge. Once addition is completed,the temperature and sparge are continued, with occasional replacement ofwater lost to evaporation, until reaction is judged to be complete byHPLC (usually 4 to 5 hours).

To remove formic acid generated during deprotection, the reactionmixture is concentrated in vacuo at no more than 40° C., until a thickviscous oil is formed. Water is added and the solution is reconcentratedto residue; this is repeated until little or no formyl proton resonanceis evident by NMR (usually after 3-4 reconcentrations), and is typicallyaccompanied by partial crystallization of DO3A as a sulfate salt.

After full dissolution in a minimum volume of water, the DO3A sulfatesalt is applied to a pretreated column of poly(4-vinylpyridine). Thetitle compound, free of sulfate, is eluted from the column withdeionized water. The aqueous solution is concentrated, and optionallylyophilized to provide the product as a hygroscopic solid.

EXAMPLE 19

Gd(III) complexes of the chelating ligands of examples 15, 16 and 17were prepared as in example 5. Purification was by standard ion exchangechromatography.

EXAMPLE 201,4,7-Tris(carboxymethyl)-10-(N-methylcarbamoylmethyl)-1,4,7,10-tetraazacyclododecane

A solution of 5 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane7.08 g (about 19 mmol assuming 5% water content) in 68 ml H₂ O wasadjusted to pH 8 using KOH. To it was added 4.4 g (40 mmol) ClCH₂CONHCH₃. The solution was warmed to 50° and the pH adjusted to 9.5 andthe pH was maintained between 9-10 by the addition of KOH as required.After 23 hours the solution was cooled to room temperature, acidified topH 3, then applied to a 500 ml bed volume of Dowex 50-X2 cation exchangeresin (H⁺ form). The column was washed with eight volumes of water thenthe product eluted with two volumes of 0.5M NH₃. Evaporation gave ayellow glassy solid. This solid was taken up in MeOH and the productprecipitated with acetone. Obtained was 3.95 g of the title compound asa slightly yellow solid.

EXAMPLE 21Gadolinium(III)(1,4,7-Tris(carboxymethyl)-10-(N-methylcarbomoylmethyl)-1,4,7,10-tetraazacyclododecane)

The pH of a mixture of 3.47 g of the crude ammonium salt from Example 20(8.33 mmol assuming 100% of the tris NH₃ salt) and 1.58 g Gd₂ O₃ (4.37mmol) in 33 ml water was adjusted to a pH of 4 using glacial aceticacid. The mixture was heated with stirring to 100° C. for 2 hoursdissolving most of the solid. The mixture was cooled and the slightamount of remaining solid removed by filtration through a 0.2 micronfilter. The filtrate was passed through a 500 ml bed of Chelex 100(ammonium form), then through a 500 ml bed column of AG1-X8 anionexchange resin (formate form). The solution was concentrated and theproduct further purified by preparative HPLC. Evaporation gave 3.1 g ofthe title compound as a colorless solid (5.2 mmol, 63% calculated for3.5% water). The complex may be recrystallized from water.

EXAMPLE 221,4,7-Triscarboxymethyl-10-(2'-hydroxypropyl)-1,4,7,10-tetraazacyclododecan

To a solution of 1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane5.19 g (14.3 mmol assuming 5% water) in 30 ml water was added 2.4 g (6.0mmol) NaOH; the pH of resulting solution was then 12.2. The solution wascooled to room temperature then 1.3 g (2.3 mmol, 1.5 equiv. ) ofpropylene oxide was added. The stoppered flask was left to stir at roomtemperature for 14 hours. HPLC analysis at that point indicated a smallamount of starting material so an additional 0.25 g (4.3 mmol, 0.28equiv.) of propylene oxide was added. After 4 hours, the reactionsolution was acidified to pH 2.9 with concentrated HCl, diluted to 0.5liters with water, then applied to a 5×40 cm column of Dowex 50X-2cation exchange resin (H⁺ form). The column was washed with six litersof water then the product was eluted with 0.5M NH₃. Obtained afterrotary evaporation was 5.9 g of the title product as the ammonium salt.

EXAMPLE 23Gadolinium(III)(1,4,7-tricarboxymethyl-10-(2'-hydroxypropyl)-1,4,7,10-tetraazacyclododecane)

To a solution of 5.6 g of the crude ammonium salt of Example 22 in 30 mlwater was added 6.70 g (16.5 mmol) of Gd(OAC)₃.4H₂ O. After 14 hours,the pH of the solution was adjusted from 4.5 to 7.0 with dilute NaOH. Asolution of 1.5 g (4.0 mmol) Na₂ EDTA in 10 ml H₂ O (pH adjusted to 7.5with dilute NaOH) was added and the resulting solution allowed to standfor 6 hours. After dilution to 0.5 liters with water the solution wasapplied to a 5×40 cm column of BioRad AG1-X8 anion exchange resin(formate form). After loading, the column was eluted with 1 liter ofwater. The total volume of eluent was collected as one fraction.Evaporation gave the title compound as a white solid. The complex wasfurther purified by preparative HPLC. Obtained was 4.6 g of a colorlesssolid (12.3% water, 7.2 mmol). The complex was recrystallized from CH₃CN.

EXAMPLE 241,4,7-Tris(carboxymethyl)-10-(2'-cyanoethyl)-1,4,7,10-tetraazacyclododecanatogadolinium

Into a 100 ml round bottom flask containing 40 ml of H₂ was placed 4.0 g(10 mmol) of crude material from example 15 and 4.5 g (11 mmol, 1.1 eq.)of Gd(OAc)₃.4H₂ O. The pH of the solution was 4.85. The mixture wasallowed to stir at room temperature for 14 hours. The reaction solutionwas then analyzed via HPLC for both free ligand and for free gadolinium.The sample was found to contain a large excess (>20%) of free metal andno detectable amount of free ligand. The pH of the solution wasincreased to 6.95 with dilute NaOH resulting in a white suspension. Thesuspension was filtered through a 0.22 micron filter and the solutionpurified via preparative HPLC. The major peak from each injection wascollected and the solution concentrated on a rotary evaporator to yield4.2 g of the gadolinium complex as a white solid. Analysis of thismaterial indicated that an unacceptable amount (5%) of free gadoliniumwas still present. The sample was dissolved in 420 ml of H₂ O and the pHof the solution adjusted to 7.5 with dilute NaOH. The solution wasapplied to a 75 ml (2.5 cm×13 cm) column bed of Chelex-100 (NH₄ ⁺ form)at a flow rate of 20 ml/min. The column was rinsed with 1 L of H₂ O andthe effluent collected and concentrated on a rotary evaporator to yielda white solid. The solid was found to contain less than 0.1% freegadolinium. However, there was a small impurity of an unidentifiedgadolinium complex. The material was subsequently repurified viapreparative HPLC. The desired peak was collected and concentrated on arotary evaporator, dissolved in anhydrous methanol and then taken todryness on a rotary evaporator and put under high vacuum for 14 hours atroom temperature to yield 3.0 g (54%) of1,4,7-tris(carboxymethyl)-10-(2'-cyanoethyl)-1,4,7,10-tetraazacyclododecanatogadolinium,as a hygroscopic white solid.

EXAMPLE 25 1,4,7-tris(carboxymethyl)-10-carbamoylmethyl1,4,7,10-tetraazacyclododecane

The pH of a solution of 1.40 g of1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane in about 4 mlwater was adjusted to 9.5 using 40% aqueous benzyltrimethylammoniumhydroxide. To the resulting solution was added 412 mg α-chloroacetamide.The temperature was increased to 80° C. and base was added as necessaryto maintain the pH at 9.5-10. After 3 hours the solution was cooled toroom temperature and acidified to pH 3 with concentrated HCl. Theresulting solution was evaporated under reduced pressure to a colorlesssludge. The mixture was taken up in about 25 ml MeOH and re-evaporated.The thick residue was triturated with a 1:1 mixture of acetone andethanol to provide a granular solid and colorless solution. The solidwas collected by filtration, washed with acetone/ethanol followed byacetone and finally ether, then dried in a vacuum oven at 50° for 2hours. Obtained was 1.54 g of the title compound as a colorless powder.The product was twice crystallized from ethanol/water.

Anal Calcd for C₁₆ H₃₁ N₅ O₇ Cl₂ +1% H₂ O: C, 39.94; H, 6.61; N, 14.55Found: C, 39.96; H, 6.74; N, 14.33

EXAMPLE 261,4,7-Tris(carboxymethyl)-10-carbamoylmethyl-1,4,7,10-tetraazacyclododecanatogadolinium

A mixture of 87 mg of1,4,7-tris(carboxymethyl)-10-carbamoylmethyl-1,4,7,10-tetraazacyclododecaneand 40 mg of Gd₂ O₃ in 0.8 ml water was heated to 80° C. for 3 hours.After cooling to room temperature, the slightly cloudy solution wasclarified by filtration through a 0.22 micron filter. The water wasremoved under reduced pressure. The residue containing the titlecompound was crystallized from a mixture of H₂ O/EtOH/CH₃ CN (1:2:4).

Anal Calcd for C₁₆ H₂₆ N₅ O₇ Gd +6.18% H₂ O: C, 32.33; H, 5.10; N, 11.78Found: C, 32.59; H, 5.10; N, 11.62; H₂ O 6.18

EXAMPLE 271,4,7-tris(carboxymethyl)-10-(4-nitro)benzyl-1,4,7,10-tetraazacyclodedecane

To a suspension of DO3A (1.02 g, 2.93 mmol) and K₂ CO₃ (1.22 g, 8.81mmol) in 10 ml DMF/H₂ O (5:3) was added a solution of4-nitrobenzylbromide (866 mg, 4.01 mmol) in 3 ml of DMF. The suspensionwas stirred at 60° C. for 24 hours which yielded a solution containing asmall amount of insoluble material. The solution was evaporated undervacuum and resuspended in 20 ml H₂ O. The suspension was acidified to pH3 with 2M HCl and extracted with 2×10 ml ethyl acetate. The aqueouslayer was evaporated under vacuum to a solid and redissolved in 50 ml H₂O. This solution was loaded onto a column (2.5×13 cm) of Dowex 50WX8cation exchange resin prepared in the acidic form. After washing thecolumn with H₂ O (ca. 500 ml), the column was eluted with 0.5M NH₄ OH(ca. 500 ml). The effluent was collected in one fraction and evaporatedunder vacuum to afford 1.35 g of crude product as the ammonium salt.

The ammonium salt (508 mg, 1.02 mmol) was dissolved in 5 ml H20 andadjusted to pH 8.4 with dilute NH₄ OH. This was placed on a column(1.5×25 cm) of AG-1X8 anion exchange resin prepared in the formate form.The column was washed with H₂ O, then the ligand was eluted with 250 mlof 0.5M HCO₂ H. The effluent was collected as one fraction andevaporated under vacuum to afford a glassy solid. This solid wasredissolved in 100 ml of H₂ O, evaporated to dryness, then crystallizedfrom 5 ml H₂ O to yield 214 mg (40.4% based on DO3A of the titlecompound as a colorless solid. The compound was pure by ¹ H and ¹³ CNMR. HPLC analysis showed trace impurities (<5% ).

Anal Calcd for C₂₁ H₃₁ N₅ O₈.12.59%H₂ O: C, 45.71; H, 6.22; N, 12.63.Found: C, 45.78, H, 7.08; N, 12.72

EXAMPLE 281,4,7-tris(carboxymethyl)-10-(4-amino)benzyl-1,4,7,10-tetraazacyclododecanatogadolinium

To a solution of1,4,7-tris(carboxymethyl)-10-(4-nitro)benzyl-1,4,7,10-tetraazacyclododecanane,(193 mg, 0.40 mmol) in 5 ml H₂ O was added solid Gd(OAc)₃.4H₂ O (219 mg,0.54 mmol). The resulting solution was stirred at 60° C. for 2 hours,then adjusted to pH 7.0 with 1.0M tris base and stirred for anadditional 1 hour. The reaction solution was diluted to 10 ml with H₂ Oand placed in a Parr bottle containing 200 mg of Raney Nickel, washed toneutral pH with H₂ O suspended in 3 ml H₂ O. The complex washydrogenated under 20 p.s.i.g H₂ for 3 hours. The catalyst was removedby centrifugation and the supernatant (pH 6.7) was filtered through a0.2 micron filter. This solution was evaporated under vacuum to afford564 mg of a solid. The solid was dissolved in 2 ml of H₂ O and placed ona column (1.0×20 cm) of Diaion CHP20P reversed phase resin packed in H₂O. After eluting the column with H₂ O (100 ml), the solvent was changedto 50% MeOH by use of a linear gradient (100 ml). Elution of the complexwas detected by UV (280 nm) and collected in one fraction. This wasevaporated under vacuum to yield 176 mg (72% based on starting ligand)of the title compound. Anal Calcd for C₂₁ H₃₀ N₅ O₆ Gd.13.84%H₂ O: C,35.87; H, 5.80; N, 9.96 Found: C, 35.56; H, 5.51; N, 10.05

EXAMPLE 291,4,7-tris(carboxymethyl)-10-(4-isothiocyanato)-benzyl-1,4,7,10-tetraazacyclododecane

To a solution of1,4,7-tris(carboxymethyl)-10-(4-amino)benzyl-1,4,7,10-tetraazacyclododecanatogadolinium(37.8 mg, 0.06 mmol) in 2 ml H₂ O was added 1.0 mL of a 104 mM (0.15mmol) solution of thiophosgene in CHCl₃. The biphasic mixture wasstirred at 40° C. for 5 minutes then at room temperature for 1 hour. Theaqueous layer was removed and evaporated under vacuum to afford 39.1 mg(96.5%) of the title compound.

This compound may be exchange labelled with ⁹⁰ Y and used directly toreact with antibodies or other proteins which contain free lysinegroups.

EXAMPLE 301,4,7-Tris(carboxymethyl)-10-(2'-carboxy)ethyl-1,4,7,10-tetraazacyclododecanatogadolinium

Into a 50 ml round bottom flask containing 10 ml of H₂ O was dissolved1.93 g (5.58 mmol) of DO3A. The pH of the solution was adjusted to 8.3with dilute NaOH. Then 0.47 g (9.0 mmol, 1.6 eq.) of acrylonitrile wasadded and the solution allowed to stir overnight at room temperature for16 hours. The solution was then taken to dryness on a rotary evaporatorand the white solid dissolved in 20 ml of 3N NaOH. The solution wasallowed to stir at 85° C. for 6 hours under nitrogen. The solution wasadjusted to pH 4.5 with 2M HCl, then applied to a 2.5×20 cm column ofDowex 50X-2 (H⁺ form). The column was eluted with 0.5 L of H₂ O and thecompound eluted off the column with 0.5 L of 0.5M NH₄ OH. The eluate wascollected as one fraction and evaporated under vacuum to a solid. Thesolid was evaporated (2 X) from 25 ml of H₂ O to yield 2.52 g of theammonium salt of the title compound.

Into a 50 ml round bottom flask containing 1.96 g of the ammonium saltdescribed above (4.3 mmol based on a diammonium salt) was added 10 ml ofH₂ O and 0.94 g of Gd₂ O₃ (2.6 mmol). The resulting suspension wasstirred at 100° C. for 6 hours. The insoluble Gd₂ O₃ was removed bycentrifugation and the solution was adjusted to pH 7 with 1M aceticacid. This solution was combined with another solution of the titlecompound prepared similarly and passed through a 1.0×30 cm column ofChelex-100 (NH₄ ⁺ form). The effluent was collected as one fraction andthe solution was concentrated to 20 ml on a rotary evaporator. Thesolution was then purified via preparative HPLC, the major peakcollected and concentrated to dryness on a rotary evaporator to yield1.99 g of1,4,7-tris(carboxymethyl)-10-(2'-carboxymethyl)-1,4,7,10-tetraazacyclododecanato-gadolinium(68% based on ligand).

EXAMPLE 311,4,7-Tris(carboxymethyl)-10-(3'-aminopropyl)-1,4,7,10-tetraazacyclodedecane

Into a 100 ml round bottom flask containing 5.2 g of DO3A dissolved in22 ml of H₂ O (pH adjusted to 8.25 with dilute NaOH) was added 1.35 mlof acrylonitrile. The reaction was allowed to stir at room temperaturefor 14 hours. After 14 hours, HPLC analysis inidcated completeconversion to1,4,7-tris(carboxymethyl)-10-(2'-cyanoethyl)-1,4,7,10-tetraazacyclododecane.The reaction mixture was taken to dryness on a rotary evaporator toyield a glassy solid. The solid was dissolved in methanol and then takento dryness on a rotary evaporator to yield 5.9 g of crude1,4,7-tris(carboxymethyl)-10-(2'cyanoethyl)-1,4,7,10-tetraazacyclododecaneas a white solid. 3.0 g of the crude1,4,7-tris(carboxymethyl)-10-(2'-cyanoethyl)-1,4,7,10-tetraazacyclododecanewas dissolved in 150 ml of H₂ O, and the solution acidified with 6 ml ofconcentrated HCl. The solution was then added to a 500 ml hydrogenationvessel containing 1.5 g of 10% Pd/C and the reaction mixturehydrogenated at 50 psi H₂ at room temperature for 14 hours. After 14hours the catalyst removed over a Celite bed and the filtrate taken todryness on a rotary evaporator. The sample was dissolved in 200 ml of H₂O and applied to a 2.5×20 cm column of Dowex 50X-2 (H⁺ form). The columnwas eluted with 4 L of 0.5 H₂ O and the material eluted off the columnwith 1.0 L of 0.5M NH₄ OH. The eluant was concentrated to dryness on arotary evaporator, the residue dissolved in methanol and taken todryness on a rotary evaporator to yield 3.8 g of the ammonium salt of1,4,7-tris(carboxymethyl)-10-(3'-amino-propyl)-1,4,7,10-tetraazacyclododecane.

EXAMPLE 321,4,7-Tris(carboxymethyl)-10-(3'-aminopropyl)-1,4,7,10-tetraazacyclododecanatogadolinium

Into a round bottom flask containing 1.0 g of the ammonium salt of1,4,7-tris(carboxymethyl)-10-(3'-aminopropyl)-1,4,7,10-tetraazacyclododecanein 5 ml of H₂ O was added 1.1 g (0.0027 mmol) of Gd(OAc)₃.4H₂ O and thereaction was allowed to stir at room temperature for 14 hours. After 14hours the pH of the solution was adjusted to 7.0 with dilute NaOH andfiltered through a 0.22 micron filter. The filtrate was then purified ona C-18 reverse-phase preparative HPLC using a 98% H₂ O and 2% CH₃ CNeluent. The major peak collected and concentrated to dryness on a rotaryevaporator to yield1,4,7-tris(carobyxmethyl)-10-(3'-amino-propyl)-1,4,7,10-tetraazacyclododecanatogadolinium.

EXAMPLE 331,4,7-Tris(carboxymethyl)-10-[N-(2-hydroxyethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecane

A solution of 5.0 g (13.3 mmol adjusted for 8.1% H₂₀) of DO3A in 50 mlof H₂ O was adjusted to pH 8.5 using 5M KOH. To this was added asolution of 3.98 g (28.9 mmol) of N-(2-hydroxyethyl)-chloroacetamide in10 ml of H₂ O. The resulting solution was adjusted to pH 9.5 and stirredat 80° C. for 24 hours. The pH was maintained at 9.5-9.7 by occasionaladdition of 5M KOH. The solution was then cooled to room temperature andadjusted to pH 3.5 using concentrated HCl . The acidic solution wasdiluted to 200 ml with H₂ O and applied to a 4.5×20 cm column of Dowex50X-2 strong cation exchange resin, H⁺ form. The column was washed with2 L of H₂ O and the material eluted off the column with 800 ml of 0.5MNH₄ OH. Rotary evaporation of the NH₄ OH fraction gave 6.5 g of thecrude ammonium salt of the title compound.

EXAMPLE 341,4,7-Tris(carboxymethyl)-10-[N-(2-hydroxyethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecanatogadolinium

To a solution of 5.0 g of the crude ammonium salt of Example 33 in 60 mlof H₂ O was added 2.04 g of Gd₂ O₃. The mixture was adjusted to pH 4using glacial acetic acid and stirred at 100° C. for 5 hours. Theresulting cloudy solution was cooled to room temperature and filteredthrough a 0.2 micron filter. The filtrate was adjusted to pH 9 withconcentrated NH₄ OH and applied to a 2.5×25 cm column of Chelex-100,ammonium form. The column was eluted with 600 ml of H₂ O. The eluant wascollected and further diluted with an additional 200 ml of H₂ O,adjusted to pH 9 using concentrated NH₄₀ H, and applied to a 2.5×30 cmcolumn of AG1-X8 (strong anion exchange resin, formate form). The columnwas eluted with 700 ml of H₂ O, and the eluate was concentrated todryness on a rotary evaporator. The product was then purified viapreparative HPLC. The major fraction collected and evaporated to drynesson a rotary evaporator. The residue was then dissolved in 25 ml of EtOHand treated with 0.5 g of activated carbon. The moisture filtered andconcentrated to dryness on a rotary evaporator to yield 3.5 g of thetitle complex as an off-white glassy solid.

What we claim is:
 1. In a method for imaging mammalian tissue whichcomprises subjecting the patient to magnetic resonance imaging whereinsaid patient has been administered orally, intrathecally orintravenously an effective amount of a contrast agent which is a stablecomplex of a paramagnetic ion of a lanthanide element and atetraazacyclo compound, the improvement wherein said complex is chargeneutral in aqueous solution.
 2. A method according the claim 1 whereinthe tetraazacyclo compound has the formula represented below ##STR24##or a pharmaceutically acceptable salt thereof, wherein Y is oxygen or##STR25## R₁ is hydrogen, alkyl having from one to five carbon atoms,arylalkyl wherein the aryl portion is phenyl or substituted phenyl,phenyl or substituted phenyl, alkoxy having from one to five carbonatoms, hydroxyalkyl wherein the alkyl portion has from one to fivecarbon atoms and having one or more hydroxy groups ##STR26## ##STR27##wherein G is NH₂, ##STR28## NHR₄, N(R₄)₂, CN, wherein R₄ is alkyl orhydroxyalkyl, ##STR29## wherein n and m are zero or an integer from oneto five, R₂ is hydrogen or alkyl, R₃ is hydrogen, hydroxyalkyl havingfrom one to five carbon atoms and having one or more hydroxy groups,alkoxy having from one to five carbon atoms, phenyl or substitutedphenyl or phenylalkyl or substituted phenylalkyl and X is chloro, bromoor iodo, wherein the term substituted phenyl refers to phenyl groupssubstituted with one, two or three halogen, hydroxyl, alkyl, alkoxycarbamoyl or carboxyl groups.
 3. The method of claim 1 wherein saidtetraazacyclo compound is a tetraazacyclododecane.